Enhanced Production Of Ginsenosides In Cell Cultures Of Panax Ginseng By Addition Of Small Molecules And A Study On Their Acting Mechanism

Panax ginseng C.A. Meyer, is a famous plant having medicine and food function. Ginsenosides are one of its principal bioactive ingredients. Currently, due to the slow growth of ginseng plants, plant cell culture technology has been explored as a potential alternative for the production of ginsenosides. However, large scale application of cell culture is still limited due to the low production of the ginsenoside. Currently, the "signal transduction engineering" approach by adopting elicitor treatment has been recognized as an effective way to improve the biosynthesis of plant secondary metabolites. Searching for effective elicitors and investigating of the signaling transduction pathways related to ginsenoside biosynthesis is helpful for understanding the mechanism of secondary metabolite biosynthesis and improving the production of useful secondary metabolites further.It was reported that, as inorganic elicitors, some heavy metal salts have profound influences on secondary metabolite biosynthesis. In P. ginseng cultures, although positive effect of some heavy metals like CuSO4and VOSO4(V4+) on the ginsenoside production was evident, the stimulating mechanism of heavy metals on the ginsenoside biosynthesis remains unclear. In another aspect, some small organic compounds always have interesting eliciting effects. As was reported,2μM N,N’-dicyclohexylcarbodiimide (DCCD) was efficient to induce plant defense response and secondary metabolites biosynthesis. However, the DCCD-stimulation strategy has not been developed and the functional mechanism of DCCD on secondary metabolite biosynthesis is still largely unknown. Also, up to now, there have been no reports on the effect of DCCD elicitation in P. ginseng cell cultures.In this dissertation, effect of different heavy metals salts on production of ginsenoside by cell cultures of Panax ginseng was investigated, and vanadate (V5+) was found the most effective. Under an optimal vanadate addition (50μM and day4), maximal ginsenoside content was499.3±7.0μg/100mg DW, which was4.4-folds that of the control. In addition, the maximal content of Rbl and Rg-group ginsenosides Rgl and Re increased by10.1,1.5and5.6times, respectively, which coincided well with increased activities of UDPG-ginsenoside Rd glucosyltransferase (UGRdGT) and protopanaxatriol biosynthetic enzyme protopanaxadiol6-hydroxylase (P6H). Meanwhile, transcription levels of ginsenoside biosynthetic genes encoding squalene synthase (SQS), squalene epoxidase (SE) and dammarenediol synthase (DS) were up-regulated while that of cycloartenol synthase (CAS) was almost unchanged by Vanadate. To provide an insight into the mechanism of vanadate elicitation further, jasmonic acid (JA) content was investigated. It was found that upon vanadate treatment, JA biosynthesis was induced, and by using JA biosynthetic inhibitor ibuprofen (Ibu), decrease of JA content and the depression of ginsenoside biosynthesis with down-regulation of sqs, se and ds gene transcription were observed. These results suggest that vanadate may induce ginsenoside biosynthesis via regulation of gene transcription and enzyme activity and JA plays an important role during this process. The effects of DCCD addition on ginsenosides biosynthesis were also studied. It was found that the optimal concentration and time of DCCD addition were10microM and on day4of cultivation. Under this condition, the maximal content of total ginsenosides was3.4mg/g DW, which was3.0-folds that of the control. Meanwhile, the enzyme activities of UGRdGT, P6H and defense enzyme phenylalanine ammonia lyase (PAL) and transcription levels of sqs, se and ds were all up-regulated. To gain a better understanding of the molecular processes underlying the elicitation, we examined the nitric oxide (NO) content. It was found that upon DCCD treatment, NO generation was up-regulated. Interestingly, up-regulated NO generation and transcription levels of sqs, se and ds with DCCD treatment were compromised by an NO biosynthetic inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), while an NO donor alone recapitulated the elicitation effect of DCCD on ginsenoside biosynthesis. These results suggest that DCCD may induce the ginsenoside biosynthesis via NO signaling in the P. ginseng cells.The inducing strategies obtained and the information about the molecular mechanism for the regulation of ginsenoside by vanadate and DCCD in P. ginseng cell culture provide some useful knowledge for further investigation and manufacturing scale production of ginsenoside. This study is also considered helpful to the manipulation of other plant secondary metabolites fermentation process.